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Neuronal cell culture in vitro
Kohoutová, Šárka ; Bařinka, Cyril (advisor) ; Pavlíček, Jiří (referee)
Neuronal cell cultures are in vitro cultures of dissociated neurons that have become an essential part of many neurobiological experiments in the last century. Cultured neurons not only allow to answer questions about their physiology under complex in vivo conditions, but also can serve as a model of neurodegenerative diseases. Neuronal cells can either be isolated directly from the nervous tissue of animals at the prenatal or adult stage of development, or they can be obtained through targeted manipulations of stem cells and secondary cell lines that lead to their neuronal differentiation. Primary neurons are considered the gold standard of neurobiological research not only because of their long tradition of cultivation, but also because primary neurons retain typical neuronal properties under in vivo conditions. There are several disadvantages associated with primary neurons, including the fact that fully differentiated neurons do not proliferate and are relatively demanding in terms of culture conditions For this reason, their role is often replaced by mitotically active secondary cell lines or stem cells. This bachelor thesis summarizes the knowledge about cell cultures used to study the functions of neuronal cells and highlights the advantages and disadvantages of their use. Key words Primary...
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3D models of brain tumors
Fišer, Ondřej ; Novák, Josef (advisor) ; Bohačiaková, Dáša (referee)
Despite intensive research, glioblastoma multiforme remains one of the tumours of the central nervous system with the worst prognosis. The ability of glioblastoma cells to infiltrate brain tissue by forming invasive microtubular structures is stimulated by contact with adjacent non- tumor cells. Intercellular communication and the influence of the extracellular matrix create a specific microenvironment that affects cell signaling, proliferation, differentiation and response to pharmaceuticals. The recurrent form of glioblastoma often displays a much faster progression than the initial disease, which is attributed to the development of resistance to therapeutics and the preservation of the proliferative capacity of some tumour cells. The discovery of the stem- cells ability to self-aggregate in suspension has led to the creation of 3D in vitro models - brain organoids. They are much more complex that the established 2D models and their heterogeneity provides an environment simulating the in vivo state. This thesis aims to describe their use in brain tumour research and techniques for culturing 3D aggregates of neural lineage formed from induced or embryonic human stem cells with respect to their gradually increasing complexity. It also presents methods of addressing issues of hypoxia, organoid...
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Analysis of pluripotent gene expression program in early embryos and embryonic stem cells
Moravec, Martin ; Svoboda, Petr (advisor) ; Motlík, Jan (referee)
Pluripotence je schopnost buňky diferencovat do jakéhokoliv buněčného typu. Formuje se během časného embryonálního vývoje u savců a její vznik je spojen s reprogramací genové exprese na globální úrovni. Proces přirozeného vzniku pluripotence není stále zcela pochopen. Pro získání nového pohledu na události, které vedou ke vzniku pluripotence u savců, studovali jsme změny v genové expresi během oocyt-zygotického přechodu u myši. V tomto modelovém systému, oplodněné vajíčko podstoupí reprogramaci, která vede k vytvoření pluripotentních blastomer. Tyto blastomery zakládají samotné embryo. Cílem mé diplomové práce bylo analyzovat aktivaci transkripce během časného vývoje a vyvinout metodu pro monitorování exprese genů v oocytech, časných embryích a embryonálních kmenových buňkách. Metoda využívá kvantitativní PCR a umožnuje změřit expresi až 48 vybraných genů, které slouží jako markery pro maternální degradaci, aktivaci pluripotentního programu a diferenciaci do zárodečných linií. Dále ukazujeme, že náš systém monitoruje dynamiku transkriptomu během oocyt-zygotického přechodu, a získané výsledky jsou srovnatelné s daty naměřenými pomocí jiných metod. Díky našemu bioinformatickému přístupu jsme navíc identifikovali nové oocyt-specifické a zygotické nekódující RNA. Klíčová slova: pluripotence,...
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